Central nervous system gliomas range in clinical behavior and histological appearance from indolent well-differentiated lesions to highly anaplastic, rapidly growing neoplasms. A cardinal property of almost all types of gliomas is a propensity to recur and undergo anaplastic change.1 The p53 tumor suppressor gene is the most frequently altered gene in human cancer and is also found mutated in several types of brain tumors.2 Mutations in the p53 tumor suppressor gene are frequently detected in gliomas. P53 is well-known for its ability to reduce cell cycle arrest, apoptosis, senescence, or differentiation following cellular stress.3 Murin-double-minute 2 (MDM-2) is an important negative regulator of P53. MDM-2 overexpression leads to inhibition of P53-mediated transactivation and enhancement of tumor progression.4 In this study, we measured the expression of P53 protein and MDM-2 protein in patients with glioma, and furthermore analyzed the association between the expression of the two proteins and the histopathological grades of glioma. We also analyzed the relationship between the expression of P53 protein and that of MDM-2 protein.
Patients and surgical specimens
Two hundred and forty-two paraffin-embedded tissues, dated from September 2009 to August 2010, including 30 normal brain tissues from patients with craniocerebral injury and 212 tissues from patients with primary glioma, were obtained from the Department of Pathology, Beijing Tiantan Hospital, Capital Medical University, China. The tumor specimens had all been taken before radiotherapy and chemotherapy were given. There were 116 men and 96 women with a mean age of 41.9 years (range 13-67 years). Histopathological assessment and grading were done according to the World Health Organization Classification of central nervous system tumors, 2000. These patients fell into three groups: control group (30 cases of craniocerebral injury), grade I-II tumor group (5 cases of grade I, 119 cases of grade II), and grade III-IV tumor group (53 cases of grade III, and 35 cases of grade IV).
Immunohistochemical staining was done using the streptavidin peroxidase conjugated method (S-P method). Briefly, the 4-μm-thick paraffin sections were de-paraffinized in dimethylbenzene, rehydrated through a graded ethanol series, and then incubated with fresh 3% hydrogen peroxide for 10 minutes at room temperature. After phosphate buffered saline (PBS) rinse, antigen retrieval from the tissue was carried out by microwaving in citrate buffer (pH 6.0) at 95°C for 10 minutes. Next, sections were incubated with 10% normal goat serum in PBS for 15 minutes at room temperature to block nonspecific binding. After rinsed with PBS, slides were incubated overnight at 4°C with mouse anti-human P53 monoclonal antibody (1:200 Neomarkers, USA) or mouse anti-human MDM-2 monoclonal antibody (1:100, Neomarkers). After PBS rinse, tissue sections were incubated for 20 minutes at room temperature with polymer enhancer (Zhongshan, China), and rinsed with PBS. Slides were then incubated for 30 minutes at room temperature with poly-horseradish peroxidase (HRP)-antihuman IgG (Zhongshan). After rinsing with PBS, slides were stained with fresh diaminobenzidine (DAB) solution, and then counterstained with Mayer's haematoxylin, dehydrated and mounted.
Evaluation of immunohistochemical staining
As a positive control for MDM-2 and P53, we used the breast carcinoma cells that showed positive signals in nuclei. As a negative control, the primary antibodies were replaced by PBS. Only the positive nuclear staining was evaluated. Assessment of both antigens was made by counting 1000 cells per section at high magnification (high-power field, 40×), and the percentage of positive tumor cells was determined. A tumor was considered positive for MDM-2 and P53 when more than 10% of the tumor cells were immunopositive. All immunohistochemistry slides were evaluated independently by two pathologists without any prior knowledge of the patients' clinical data. When the opinions of the two pathologists were different, agreement was reached by careful discussion.
All statistical analyses were conducted using SPSS for windows version 17.0 (SPSS Inc., USA). In immunohistochemical study, relationship between the expressions of P53 protein and MDM-2 protein and pathological grades were analyzed by chi-square test. P <0.05 was considered statistically significant. Division was performed when the difference between any two groups was analyzed, and the corrected α=0.017, namely P <0.017 was considered as statistically significant in analyzing the difference between any two groups. The relationship between MDM-2 protein expression and P53 protein expression was established by the Kappa test.
Expression of P53 protein
All 30 tissues of the control group were P53 negative. Sixty-six of 124 glioma cases of grade I-II were P53 positive, a P53 positive rate being 53.2%; 66 of 88 glioma cases of grade III-IV were P53 positive, a P53 positive rate being 75.0%. The P53 positive rate was significantly higher in the glioma groups than in the control group (χ2=27.944, P <0.0001; χ2 =51.058, P <0.0001). The P53 positive rate was significantly higher in glioma tissues of grade III-IV than that in glioma tissues of grade I-II (χ2 =10.386, P=0.001) (Table 1, Figure 1).
Expression of MDM-2 protein
All 30 tissues from the control group were MDM-2 negative. Ninety-nine of 124 glioma cases of grade I-II were MDM-2 positive, and the MDM-2 positive rate was 76.6%; 67 of 88 glioma cases of grade III-IV were MDM-2 positive, and the MDM-2 positive rate was 76.1%. The MDM-2 positive rate was significantly higher in the glioma groups than in the control group (χ2 =59.992, P <0.0001; χ2 = 52.848, P < 0.0001). There was no significant difference in MDM-2 expression between the two glioma groups (χ2 =0.006, P=0.936) (Table 2, Figure 2).
Relationship between the expression of P53 protein and that of MDM-2 protein
The expression of P53 protein was not related to that of MDM-2 protein (Kappa value=0.153, P=0.069) (Table 3).
The p53 gene, located on chromosome 17p, encodes a 53-kDa nuclear phosphoprotein, which can bind to DNA and act as a transcription factor. Normal p53 (wild-type p53) is believed to function as an inhibitor of cell replication when DNA damage is sustained. It is generally undetectable by standard immunohistochemistry because of its low cellular levels and very short half-life.5 Mutations of the gene occurring within the coding region usually lead to the production of a non-functional protein (mutant-type P53) which, being much more stable than the wild-type protein, accumulates in the nucleus reaching the threshold of immunohistochemical detection.6 Analysis of human cancers reveals a fundamental role for P53 in tumor suppression. More than half of human cancers, a wide variety of types, harbor p53 mutations.7 In our study, the glioma groups exhibited higher levels of P53 protein than the control group, and the P53 positive rate was significantly higher in the grade III-IV glioma group than in the grade I-II glioma group. The positive rates of P53 protein are gradually increased with the increase in the histological grade of the glioma. This may imply that overexpression of P53 protein might be related to the occurrence and progression of glioma. This result is consistent with that by Ranuncolo et al.8
The MDM-2 gene, originally identified as being gene-amplified on double-minute chromosomes in transformed mouse fibroblasts, located on chromosome 12, is considered a negative regulator of P53 function and seems to play a role in the pathogenesis of a variety of tumors.9 Recent studies have shown that MDM-2 is activated in response to a variety of oncogenic pathways independent of P53. Although its role as an oncogene via suppression of P53 function remains unclear, growing evidence argues for P53-independent effects, as well as the possibility that MDM-2 has tumor suppressor functions in the appropriate context. Hence, MDM-2 is proving to be a key player in human cancer in its own right, and thus an important target for therapeutic intervention.10 There was no agreed viewpoint on the association between the expression of MDM-2 protein and the histological grade of glioma.11,12 Our study found that glioma groups exhibited higher levels of MDM-2 protein than the control group, but there was no statistical difference in levels of MDM-2 protein between the two glioma groups. This suggested that overexpression of MDM-2 protein may play an important role in glioma tumorigenesis, but may not be involved in glioma progression. The overexpression of MDM-2 protein was an early event in malignant transformation of glioma. Our results are in accordance with those found by Kamiya et al,12 but contradictory results were obtained by some studies.13,14 Our immunohistochemical study showed that overexpression of P53 protein did not depend on MDM-2 protein overexpression, suggesting that MDM-2 may be a key player in glioma in its own right, and this viewpoint is supported by other evidence.10
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Keywords:© 2011 Chinese Medical Association
P53 protein; MDM-2 protein; glioma